U.S. patent number 5,677,176 [Application Number 08/404,372] was granted by the patent office on 1997-10-14 for animal derived cell with antigenic protein introduced therein.
This patent grant is currently assigned to Hapgood, C.V.. Invention is credited to Garret M. Ihler, Yves-Claude Nicolau.
United States Patent |
5,677,176 |
Nicolau , et al. |
October 14, 1997 |
Animal derived cell with antigenic protein introduced therein
Abstract
This invention comprises animal derived cells, especially
erythrocytes, which have been artificially modified (and referred
to as engineered erythrocytes or RBCs) so as to incorporate in
their plasma membranes an antigen, CD4 protein derived from
lymphocytes) which will cause them to selectively seek out and fuse
with other cells that are infected with a virus, especially the
AIDS virus. These modified cells can be further altered so as to
contain in their cytoplasm a cytotoxic agent which, after the cell
has fused with a target cell, will result in the pooling of their
respective cytoplasms and the death of both cells. Such modified
cells can be used as a basis for an in vitro diagnostic assay
involving cells derived from the blood of patients suspected of
having AIDS.
Inventors: |
Nicolau; Yves-Claude (College
Station, TX), Ihler; Garret M. (College Station, TX) |
Assignee: |
Hapgood, C.V. (Oldwick,
NJ)
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Family
ID: |
26748809 |
Appl.
No.: |
08/404,372 |
Filed: |
March 15, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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46384 |
Apr 9, 1993 |
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771874 |
Oct 2, 1991 |
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197445 |
May 27, 1988 |
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68288 |
Jun 30, 1987 |
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Current U.S.
Class: |
435/325;
530/350 |
Current CPC
Class: |
A61P
43/00 (20180101); A61K 35/18 (20130101); A61K
9/5068 (20130101); B82Y 5/00 (20130101); G01N
33/5432 (20130101); A61K 9/1271 (20130101); A61K
38/168 (20130101); A61P 31/12 (20180101); G01N
33/555 (20130101); G01N 33/56988 (20130101) |
Current International
Class: |
A61K
35/18 (20060101); A61K 38/16 (20060101); C12N
005/06 (); C07K 014/00 () |
Field of
Search: |
;435/173.4,71.3,240.1,325 ;530/350 ;536/23.5 |
References Cited
[Referenced By]
U.S. Patent Documents
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5236835 |
August 1993 |
Mouneimne et al. |
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Other References
Doxsey et al. An efficient method for introducing macromolecules
into living cells J. Cell Biol. vol. 101 19-27 1985. .
Maddon et al. The isolation and nucleotide sequence of a cDNA
encoding the T cell surface protein T4: A new member of the
immunoglobulin gene family Cell vol. 42 93-104 1985. .
Gad et al. Fusion of cells and proteoliposomes: Incorporation of
beef heart cytochrome oxidase into rabbit erythrocytes FEBS Lett.
vol. 102 230-234 1979. .
Mayhew et al. Therapeutic applications of liposomes Chapter 7 in
Liposomes M.J. Ostro Ed. Marcel Dekker, New York and Basel. 1983.
.
Dimitriadis et al. Liposome-mediated ricin toxicity in
ricin-resistant cells FEBS Lett. vol. 98 33-36 1979. .
Yarchoan et al. Administration of 3'-azido-3'-deoxythymidine, an
inhibitor of HTLV-III/LAV replication, to patients with AIDS or
AIDS-related complex Lancet 575-580 Mar. 15, 1986..
|
Primary Examiner: Ketter; James
Assistant Examiner: Brusca; John S.
Attorney, Agent or Firm: Baker & Botts, L.L.P.
Parent Case Text
This application is a continuation of U.S. application Ser. No.
08/046,384, filed Apr. 9, 1993, now abandoned, which is a
continuation of U.S. application Ser. No. 07/771,874, filed Oct. 2,
1991, now abandoned, which is a continuation of U.S. application
Ser. No. 07/197,445, filed May 27, 1988, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 07/068,288, filed
Jun. 30, 1987, now abandoned.
Claims
What is claimed is:
1. A mammalian-derived red blood cell containing a recombinant
human CD4 protein inserted into its cellular membrane which induces
the mammalian-derived red blood cell to selectively bind to and
fuse with a target, wherein the mammalian-derived red blood cell's
life span is about a normal red blood cell's life span and wherein
the recombinant human CD4 protein is non-immunogenic.
2. The mammalian derived red blood cell according to claim 1,
further comprising one or more cytotoxic agents incorporated
therein.
3. The mammalian derived red blood cell according to claim 2,
wherein the cytotoxic agent is a protein.
4. The mammalian derived red blood cell according to claim 2,
wherein the cytotoxic agent is selected from the group consisting
of ricin, abrin, gelonin, and diphtheria toxin and toxicologically
active fragments thereof.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an animal derived cell into whose
membrane is introduced an antigenic protein which predisposes the
cell to bind and fuse with other cells. More particularly, the
present invention concerns modified cells and liposomes, wherein
their outer membranes have incorporated therein specific protein
substances which will cause the modified cells or liposomes to bind
selectively, in vivo and in vitro, to various target cells,
especially those infected with a virus, e.g., the human
immunodeficiency virus (hereafter HIV).
2. Background Information
It is known that Acquired Immune Deficiency Syndrome (AIDS) is a
virulent disease characterized by a well defined chronological
sequence of symptoms and a high rate of mortality (Curran, J. W. et
al, The Epidemiology of AIDS, Science, 229, 1352-1357 (1985)). AIDS
was first described in 1981 and since that time has reached
epidemic proportions with some 400,000 cases in the United States
alone and a 3 year mortality rate of over 90%. It is now estimated
that approximately 1 million people in this country have been
infected with the human immunodeficiency virus (HIV). HIV is
classified as a retrovirus containing core proteins, genomic RNA,
and the enzyme reverse transcriptase. Antibodies to several
antigens of HIV are present in the serum of infected persons.
The hallmark of the immunodeficiency in AIDS is depletion of the T4
helper/inducer lymphocytes (Gottlieb et al, N. Engl. J. Med. 305,
1425-1431 (1981)). This defect is primarily the result of selective
infection by HIV of this population of lymphocytes. The T4 molecule
(denoted CD4-antigen) present on the surface of helper lymphocytes
has been implicated as the receptor for the HIV virus (Dalgleish et
al, Nature, 312, 763-767 (1984)) which enters the cell after
specific binding to the surface of the T4 lymphocyte. The mechanism
of viral entry has not been completely defined but it may be
similar to either receptor-mediated endocytosis or direct fusion of
the HIV envelope with the cell membrane (Stein et al, Cell 49,
659-668 (1987)).
A fusogenic protein, called gp120 (a glycoprotein of molecular
weight 120,000 daltons) (McDougall et al, Science, 231, 382-385
(1986)) has been identified on the HIV surface and this protein may
serve to mediate fusion between the virus and the lymphocyte. Once
inside the cell, the viral RNA is transcribed into DNA by reverse
transcriptase. Subsequently, the DNA is integrated into the host
genome. However, most of the HIV DNA remains unintegrated and in
the cytoplasm. It is now known that HIV replication is restricted
at this stage until the infected cell is "activated" (McDougal et
al, J Immunology, 135, 3151-3162 (1985)). It is believed that the
potential activators of replication, following HIV infection,
include viruses such as hepatitis B, human cytomegalovirus, and
herpes simplex virus. Upon activation, the HIV is replicated and
then assembled on the cell surface. Mature virions are then formed
by budding from the surface membrane of the T4 lymphocyte.
Subsequent to initiation of HIV replication, the T4 lymphocyte may
be killed.
While the cytopathic effect of HIV virus for T4 lymphocytes is
presently unknown, it has been observed that when HIV infection
occurs, the viral gp120 antigen is expressed on the surface of the
infected T4 lymphocyte. Because of the affinity of this protein for
the normally present CD4 antigen, other T4 lymphocytes (uninfected
and possessing the CD4 antigen) can fuse with the infected
lymphocyte. The resulting binding and fusing of the cells appears
to kill both, or all, off the cells involved in the fusion (Zagury
et al, Science, 231, 850-853 (1986)). For example, T4 lymphocytes
from cell lines which lack the CD4 antigen on their surfaces as
well as T4 cells in which the antigen has been masked by reaction
of the cells with anti-CD4 antibodies do not demonstrate fusion
with HIV-infected T4 cells under the usual conditions (McDougall,
supra).
This fusion process can involve a few HIV-infected and a large
number of noninfected T4 cells and could lead to the formation of
large syncytia which could then either be removed from the
circulation by the cells of the reticuloendothelial system or else
lyse (e.g., in organs like the brain) (Shaw et al, Science, 227,
177-181 (1985); Gartner et al, Science, 233, 215-219 (1986)).
The T4 lymphocyte plays a central role in the immune response. It
is intimately involved with macrophages, cytotoxic T cells, NK
(natural killer) cells and B lymphocytes. Therefore, even a
selective depletion of the T4 lymphocyte population can result in a
multitude of immunologic defects leading to the life-threatening
opportunistic infection characteristic of AIDS (Bowen et al., Ann
Intern Med, 103, 704-709 (1985)). In addition, certain populations
of monocytes and macrophages also express the CD4 antigen and
studies have shown that these cells can also become infected with
HIV (Ho et al, J Clin Invest, 77, 1712-1715 (1986)). HIV infection
of monocytes can result in a defect in chemotaxis which has been
reported in AIDS. The infected macrophages may carry the HIV virus
into the central nervous system allowing for the development of the
subacute encephalitis that occurs in this disease (Gabuzda et al,
Ann Neurology, 20, 289-295 (1986)). HIV infected monocytes may
produce a variety of factors, including tumor necrosis factor, that
could explain the chronic fever of AIDS and also the associated
condition of cachexia (general malnutrition).
Furthermore, B lymphocyte abnormalities, consisting of polyclonal
activation with high levels of immunoglobulin coupled with poor
antibody response to new antigens are common with AIDS and may be a
direct consequence of the HIV infection. Patients in the more
advanced stages of AIDS are usually anergic (i.e., exhibit
diminished immunological response to common antigens).
AIDS has proven extremely difficult to treat, let alone cure.
Heretofore, several different approaches have been tried. Among the
potential treatments currently being investigated are the
following:
a. Inhibitors of reverse transcriptase. This type of treatment can
be highly selective because the target enzyme is not found in human
cells. The prototype drug in this class is azido-3'-deoxythymidine
(AZT). This drug has passed through phase I, II, and III studies
and is presently approved for treating patients with AIDS and who
have had previous pneumocystis infection. Studies have shown that
patients treated with this drug exhibit increased levels of helper
T lymphocytes and about a third demonstrate positive skin test
(where the latter were previously anergic). In addition, there is
an improvement over the neurological deficiencies characteristic of
the disease (see Yarchoan et al, Lancet, pp 575-580 (1986)).
However, despite the aforestated improvements in clinical and
immunological parameters, the virus is found to persist in
lymphocytes.
b. General antiviral agents. Currently under investigation are such
compounds as ribavirin, Foscarnet (HPA-23), and suramin. No data is
presently available on the effectiveness of these agents.
c. Immunomodulators. This type of treatment involves attempts to
enhance or reconstitute the defective immune system in patients
with AIDS. Such trials have been taking place for several years.
Among the immunomodulators being examined are Alpha Interferon, a
leukocyte-derived glycoprotein possessing antiviral
immunoregulatory and anti-proliferative effects. This agent has
exhibited only minimal effectiveness in human patients while
showing unacceptable toxicity levels (Celmann et al, Am J Med, 78,
737-741 (1985)). A similar agent tested in AIDS patients is
Interieukin-2. The latter has been shown to elevate the total
number of T-lymphocytes, and to decrease, but not eliminate, the
isolation of HIV from lymphocytes. It has also been implicated in
minor degrees of regression of Kaposi's sarcoma, the latter a
malignant tumor associated with the more advanced stages of AIDS
(Broder et al, Lancet, pp 627-630 (1985)).
d. Transplantation. Bone marrow transplantation has been attempted
in several AIDS patients, the purpose being to reconstitute their
immunologic reactivity. Such therapy has resulted only in
transient, rather than long lasting, improvement in the
condition.
Methods of efficiently fusing liposomes with either cells or
nuclear envelopes have been described (see Arvinte et al,
Biochemistry, 26, 765-772 (1986)). In most instances such fusions
have been achieved using inducing agents, called fusogens, which
may be proteins, peptides, polyethylene glycol, viral envelope
proteins, etc. In some cases the fusion inducing agent involves
altered conditions of the medium. Thus, lowered pH has been used
advantageously to induce fusion of liposomes with nuclear envelopes
(Arvinte et al, Biochemistry, supra).
Procedures have been previously developed for the targeted delivery
of different molecules to specific cells in vivo (Wicolau et al,
Biochim. Biophys. Acta, 805, 354-367 (1984)). Such targeting was
realized by using liposomes which contained specifically selected
glycolipids in their bilayers. Such glycolipids were selected based
upon the presence on them of a terminal carbohydrate portion which
was recognized by one or more lectins (substances derived from
plant cells which bind specifically to certain types of
carbohydrate structures) on the target cell's plasma membrane (see
Wicolau et al, Proc Natl Acad Sci, 80, 7128-7132 (1983)). For such
a procedure will work, the target cell must contain a receptor in
its membrane which is specific for, and will bind to, a molecule
present in the membrane of another cell. It is then necessary to
induce the cells to fuse which can be accomplished experimentally
using fusogenic agents, or, in nature, by certain fusing agents
such as proteins derived from viral infection (Gallo et al.,
Science, 224, 500-503 (1983)).
SUMMARY OF THE INVENTION
It is an object of this invention to take advantage of the
selective fusion of HIV-infected cells with CD4-bearing cells by
producing CD4-bearing cells, advantageously erythrocytes, and
alternatively, liposomes, into which have been incorporated various
cellular toxins and lytic agents, advantageously the protein ricin.
Such a procedure results in the selective killing of HIV-infected
cells. Such object is realized by the construction of a family of
engineered red blood cells or liposomes carrying the CD4 antigen in
the plasma membrane or lipid bilayer (as the case may be) and
containing a cytotoxic agent such as ricin, gelonin, and/or
equivalents thereof.
It is a further object of this invention to present procedures for
the introduction of antigens into the plasma membranes of
cells.
It is also an object of this invention to present procedures for
the incorporation of toxic and cytolytic agents into liposomes and
cells of choice.
It is another object of this invention to alleviate viral disease
conditions by the introduction into a patient so afflicted an
optimal quantity of such engineered cells or liposomes and allowing
these cells or liposomes to bind to, fuse with an destroy viral
infected cells in vivo, before the virus can either replicate in
the infected target cell or facilitate that cell's binding to other
healthy cells so as to spread the virus or retard the body's
defenses against it.
It is also an object of this invention to present a procedure for
the administration of heterologous red blood cells (and/or
liposomes) altered by insertion of the CD4 protein into their
membranes with resultant selective binding of these cells (and/or
liposomes) to cells infected with HIV and concomitant fusion and
elimination from the circulation of the HIV infected cells.
These and other objects, aims and advantages are realized in
accordance with the present invention.
The present invention concerns an animal, e.g., human, derived cell
into whose membrane has been introduced, e.g., artificially
incorporated, an antigenic protein which predisposes the cell to
bind to and fuse with other cells.
The present invention also relates to a treatment of a virus
induced disease which comprises administering, e.g., injecting,
thereof a therapeutically effective amount of the aforementioned
animal derived cells in conjunction with one or more cytotoxic
agents, suspended in a pharmacologically acceptable diluent.
The present invention is also directed to a composition comprising
a therapeutically effective amount of the aforesaid animal derived
cells, in conjunction with one or more cytotoxic agents, suspended
in a pharmacologically acceptable diluent.
DETAILED DESCRIPTION OF THE INVENTION
a. Nature of the Invention
The present invention embodies a type of engineered red blood cell
which has incorporated into its plasma membrane the human CD4
antigen and contains within the cell one or more cytotoxic agents
capable of destroying any cell which fuses with the engineered
cell. In place of these engineered red blood cells, liposomes can
be produced and used in a similar manner. The invention also
relates to a procedure for producing these cells from normal red
blood cells. The invention further embodies a method for using
these engineered red blood cells (or liposomes) to treat diseases
caused by viral infection, especially Acquired Immune Deficiency
Syndrome (AIDS). It should be kept in mind, of course, that
whenever engineered red blood cells are referred to, the modified
liposomes described herein can also be used.
The major feature of the proposed treatment is as follows. Red
blood cells carrying on their membrane the CD4 receptors are
capable of binding to circulating HIV-infected cells expressing the
viral gp120 surface glycoprotein. Such aggregates are then removed
from the circulation by splenic macrophages and the Kupffer cells
of the liver.
In order to prevent the possible infection of these phagocytic
cells upon uptake of the cellular aggregates, certain anti-HIV
drugs such as AZT- or DDC-triphosphate (where AZT is
azido-3'-deoxythymidine and DDC is dideoxycytidine) are
encapsulated in CD4-bearing erythrocytes prior to injection.
Although phagocytosis without drugs effectively leads to the
destruction of the HIV-infected cells, additional protection
results by incorporating such anti-HIV drugs in the red cells.
Because the HIV-infected cells tend to selectively bind and
eventually fuse with noninfected cells having the CD4 antigen we
postulated and proved that any cells, even erythrocytes, possessing
this antigen on their plasma membranes would selectively bind and
possibly fuse with HIV-infected T-cells and/or with the HIV virus
itself. When an HIV-infected cell fuses with another cell
containing a cytolytic agent the infected cell is destroyed.
To be clinically effective as an agent in the destruction of virus
infected cells the engineered cells must have the following
properties: 1. they must be long lasting, i.e., have a lifespan at
least approaching the normal lifespan of the cell, in this case
erythrocytes, in vivo; 2. they must be of low toxicity so that the
cytotoxic agent contained in them will not act indiscriminately on
the tissues of the body; 3. they must not themselves be adversely
affected by the toxin placed within them (otherwise they would be
poisoned before they could seek out and selectively fuse with the
target cell); 4. they must also be selective in fusing only with
the target (i.e. infected) cell and neither bind to nor fuse with
other healthy cells; 5. they must be non-immunogenic so that they
will not cause an adverse antigenic reaction in the recipient and
thus further tax the patient's already stressed immune system. The
modified erythrocytes (and liposomes) comprising the present
invention have been discovered to be good candidates for such a
role. This is because they lack any of the vital reproductive
apparatus of other cells and, during their life-spans, are
essentially nothing more than bags of hemoglobin which serve to
carry oxygen in the blood stream. Thus, they are not themselves
adversely effected by the presence within them of various cytotoxic
agents. Since erythrocytes are among the most numerous cells in the
bloodstream the replacement of a small portion of them by
antigenically-modified toxin-laden cells is of little consequence
to the organism. Additionally, because the toxin is sequestered
within the modified erythrocytes it is not free to interact
randomly with the tissues of the organism as would be the case with
a more injected therapeutic agent.
b. Liposomes
Consist of spheres of lipid bilayers (two-molecules thick) that
enclose an aqueous medium.
Liposomes can generally be formed by sonicating a lipid in an
aqueous medium, by resuspension of dried lipid layers in a buffer
or by dialysis of lipids dissolved in an organic solvent against a
buffer of choice.
Phospholipids form closed, fluid-filled spheres when they are mixed
with water in part because the molecules are amphipathic: they have
a hydrophobic (water-insoluble) tail and a hydrophilic
(water-soluble), or "polar," head. Two fatty acid chains, each
containing from 10 to 24 carbon atoms, make up the hydrophobic tail
of most naturally occurring phospholipid molecules. Phosphoric acid
bound to any of several water-soluble molecules composes the
hydrophilic head. When a high enough concentration of phospholipids
is mixed with water, the hydrophobic tails spontaneously align
together to exclude water, whereas the hydrophilic heads bind to
water.
The result is a bilayer in which the fatty acid tails point into
the membrane's interior and the polar head groups point outward.
The polar groups at one surface of the membrane point toward the
liposome's interior and those at the other surface point toward the
external environment. It is this remarkable reactivity of
phospholipids with water that enables workers to load medications
into liposomes. As a liposome forms, any water soluble molecules
that have been added to the water are incorporated into the aqueous
spaces in the interior of the spheres, whereas any lipid soluble
molecules added to the solvent during vesicle formation are
incorporated into the lipid bilayer.
Liposomes employed for drug delivery typically range in diameter
from 250 angstrom units to several microns (for comparison, the
diameter of an erythrocyte is about 10 microns) and are usually
suspended in a solution. They have two standard forms:
"onion-skinned" multilamellar vesicles (MLV's), made up of several
lipid bilayers separated by fluid, and unilamellar vesicles,
consisting of a single bilayer surrounding an entirely fluid core.
The unilamellar vesicles are typically characterized as being small
(SUV's) or large (LUV's).
Under appropriate circumstances liposomes can adsorb to almost any
cell type. Once they have adsorbed the spheres, liposomes may be
endocytosed, or swallowed up, by some cells. Adsorbed liposomes can
also exchange lipids with cell membranes and may at times be able
to fuse with cells. When fusion takes place, the liposomal membrane
is integrated into the cell membrane and the aqueous contents of
the liposome merge with the fluid in the cell.
The ability of liposomes to adsorb, bind and eventually be taken up
by many types of cells and then slowly release their contents makes
them excellent candidates for time-release drug-delivery systems.
How quickly a drug is released from a liposome depends on numerous
factors, including the composition of the liposome, the type of
drug encapsulated and the nature of the cell.
Endocytosis of liposomes occurs in a limited class of cells, viz.,
those able to ingest foreign particles. When phagocytic cells take
up liposomes, the cells move the spheres into subcellular
organelles known as lysosomes, where the liposomal membranes are
believed to be degraded. From the lysosome, the liposomal lipid
components probably migrate outward to become part of the cell's
membranes and other liposomal components that resist lysosomal
degradation (such as certain medications) may enter the
cytoplasm.
Lipid exchange involves the transfer of individual lipid molecules
from the liposome into the plasma membrane (and vice versa); the
aqueous contents of the liposome do not enter the cell. For lipid
exchange to take place the liposomal lipid must have a particular
chemistry in relation to the target cell. Once a liposomal lipid
joins the cell membrane it can either remain in the membrane for a
long time or be redistributed to a variety of intracellular
membranes. If a drug was somehow bound to such an exchangeable
lipid, it could potentially enter the cell during lipid
exchange.
c. Diseases
The present invention can be used to combat various viral,
bacterial, allergen and parasitic diseases of man and animals.
Accordingly, the present invention can be used to combat the
following viruses: HIV, hepatitis B virus, influenze hemagglutinin
(A/memphis/102/72 sttain, A/Eng 1878/69 strain, A/NT/60/68/29c
strain, and A/Qu/7/70 strain, Ao/PR8/34, A1/CAM/46, and
A2/Singapore/1/57; Type B influenze viruses, e.g. B/Lee 40), fowl
plague virus hemagglutinin, vaccinia, polio, rubella,
cytomegalovirus, small pox, herpes simplex types 1 and 2, yellow
fever, Infectious ectromelia virus, Cowpox virus, Infectious bovine
rhinotracheitis virus, Equine rhino-pneumonitis (equine abortion)
virus, Malignant catarrh virus of cattle, Feline rhinotracheitis
virus, Canine Herpes virus, Epstein-Barr virus (associated with
infectious mononucleosis and Burkitt lymphoma), Marek's disease
virus, Sheep pulmonary adenomatosis (Jaagziekte) virus,
Cytomegaloviruses, Adenovirus group, Human papilloma virus, Feline
panleucopaenia virus, Mink enteritis virus, Infectious pancreatic
necrosis virus of trout, Fowl sarcoma virus (various strains),
Avian leukosis virus (visceral, erythroblastic and myeloblastic),
Osteopetrosis virus, Newcastle disease virus, Parainfluenze viruses
1, 2, 3, and 4, Mumps virus, Turkey virus, CANADA/58, Canine
distemper virus, Measles virus, Respiratory syncytial virus, e.g.,
B influenze viruses, e.g., B/Lec/40; Rabies virus; Eastern equine
encephalitis virus; Venezuelan equine encephalitis virus; Western
equine encephalitis virus; Yellow fever virus, Dengue type 1 virus
(=type 6), Dengue type 2 virus (=type 5), Dengue type 3 virus,
Dengue type 4 virus; Japanese encephalitis virus; Kyasanur forest
virus; Louping ill virus; Murray Valley encephalitis virus; Omsk
hemorrhagic fever virus (types I and II); St. Louis encephalitis
virus; Human rhinoviruses; Foot-and-mouth disease virus; Poliovirus
type 1; Enterovirus Polio 2; Enterovirus Polio 3; Avian infectious
bronchitis virus; Transmissible gastro-enteritis virus of swine;
Lymphocytic choriomeningitis virus; Lassa virus; Machupo virus;
Pichinde virus; Tacaribe virus; Papillomavirus; Sindbis virus; and
the like.
The present invention can also be used to combat bacteria such as
those causing leprosy, tuberculosis, syphilis and gonorrhea.
The present invention can also be used to combat parasites, for
example, organisms carrying malaria (Plasmodium falciparum, P.
ovale, etc.), Schistosomiasis, Onchcerca volvulus and other
filiarial parasites, Trypanosomes, Leishmania, Chargas disease,
amoebiasis, hookworm, and the like.
Since the present invention permits targeting of the modified cells
and liposomes to particular cells and tissues, it can also be
effective in combating cancerous growths.
e. Production of Pure CD4
The destruction of normal T-cells by HIV involves the infectiono f
the cell by the virus with subsequent production of specific
glycoproteins coded for by the virus and insertion of these
glycoproteins into the plasma membrane of the infected cell. This
glycoprotein has an affinity for other cells which contain the CD4
antigen on their surfaces. The human leukocyte antigen, CD4 can be
isolated from various sources including the buffy coat obtained
following centrifugation of blood from a blood bank as well as from
a T-cell lymphoma cell line (CEM-cells obtained from the American
Type Culture Collection, Rockville, Md., USA). A CD4 antigen can be
purified by the procedure of Maddon et al., Cell, 42, 93-104
(1985).
A general scheme for this procedure is shown hereinbelow as
follows: ##STR1##
Purified CD4 is ready for use and is stable for 2-3 months at
-20.degree. C. In the above scheme, the buffer compositions could
be as follows:
Buffer A=0.2M n-octyl-.beta.-D-galactoside (OGS) 0.15M NaCl 0.2M
PMSF 0.01M Tris, final pH=8.0.
Buffer B=Buffer A containing 0.1M beta-mannoside
Buffer C=1% (w/w) Sodium deoxycholate 1M Sodium Acetate, final
pH=4.0.
Buffer D=0.1M Acetate, pH=4.7.
In Buffer A, above, PMSF (phenyl methyl fluorosulfonic acid) is a
toxic substance used to inhibit proteolysis during the extraction
and purification procedure. However, it is completely removed by
the subsequent chromatography steps.
OGS (octyl-galactoside) is a detergent used in the extraction of
CD4. Later in the purification process, it is replaced by DOC
(deoxycholate), which is a naturally occurring bile salt and is not
toxic.
DOC (sodium deoxycholate) will be present in purified CD4 at a
concentration of 0.005% and in the blood of AIDS patients treated
by the present invention at a concentration of about 0.000001%
(which is completely safe).
Human CD4 protein can also be obtained as a recombinant CD4
molecule in a variety of cells (Walton et al, Cell, 1988, in
press.
f. Cell Fusion to Form Engineered Red Blood Cells
A general procedure for formation of engineered erythrocytes
according to the present invention is outlined hereinbelow as
follows: ##STR2##
The modified-RBC's (red blood cells) produced in the above scheme
(unlabeled) contain about 50,000 molecules of CD4 per cell and are
stable at 20.degree. C. for up to 20 days.
The chromium labeled modified RBC's were used for toxicity studies
only, so that their production is optional and they should normally
not be used in the actual practice of this invention for
therapeutic purposes, although they could be. Such labelled cells
would find use in in vitro diagnostic procedures using the present
invention as the active reagent therein.
An alternative procedure for insertion of CD4 into erythrocyte
membranes is to first insert the protein into liposome membranes
and then fuse them with erythrocytes to yield a cell-liposome
hybrid containing CD4 in the membrane. In addition, this procedure
results in the delivery of a liposome-encapsulated molecule
(advantageously, a cytotoxin or a therapeutic agent) to a cell,
advantageously an erythrocyte, with subsequent fusion.
Techniques for the continuous lysis and resealing of erythrocytes
have been developed, i.e., based on the already known concept of
resealed red cells (Ihler et al, PNAS, 70, 2663-2666 (1973)), and
cells loaded by endocytosis (Ihler et al, J. App. Biochem., 4,
418-435 (1982)). These procedures permit the encapsulation of a
wide variety of molecules into cells while keeping their life-spans
unchanged (Nicolau et al., EP 83 401364-1 (1983) and Nicolau et al,
Ann. N.Y. Acad. Sci., 445, 304-315 (1985). The present invention
involves a modified erythrocyte (or liposome) which can act as a
"targeted bullet" for ultimate fusion with, and destruction of,
virus infected cells in vivo.
It has already been shown that lysozyme will induce fusion of
liposomes with erythrocyte ghosts at acidic pH (Arvinte et al.,
Proc. Nat. Acad. Sci., Vol. 83, 962-966 (1986)). In that procedure
the lysozyme was covalently bound to the outer surface of sonicated
vesicles (liposomes) and served to induce fusion of these vesicles
with human erythrocyte ghosts. A strong induction of fusion was
found at the lysozyme pH optimum (which was not observed when
lysozyme was merely added to the suspension). This procedure is
useful because the lysozyme does not induce fusion of electrically
neutral liposomes with each other and thus is well suited for
fusing liposomes with cells.
The present invention involves procedures for inducing fusion
without lysozyme being present in the medium at all. This greatly
facilitates the formation of engineered erythrocytes for large
scale clinical use.
g. Toxicity of Engineered Erythrocytes
The lifespan of engineered red blood cells (containing different
entrapped substances, such as inositol hexaphosphate
isothiocyanate-labeled ricin) has been shown to be very nearly the
normal value (Nicolau et al, Ann. N.Y. Acad. Sci., supra). In
addition, the in vivo toxicity of these cells over a period of 30
days in piglets has been followed. Briefly, after injection of a
small quantity of the engineered cells (about 0.1 to 1 ml of 30 to
40% drug-laden cells, i.e. the hematocrit of the cell suspension is
30 to 40%, injected intravenously) samples of the animal's blood,
e.g., piglet's blood, are withdrawn at intervals of time over the
course of thirty days. These are assayed for levels of ions
(including K, Na, Cl, Ca, etc) as well as protein, urea and glucose
levels.
Life span measurements of mouse RBC's encapsulating gelonin have
shown no significant change as compared with the lifespan of normal
mouse RBC's (both having a half life of about 11 days in mice).
During toxicity experiments, animals are sacrificed at various time
intervals over a thirty day period and the state of the liver
Kupffer cells and splenic macrophages is examined. Direct
innoculation of free immunotoxin (where ricin was the toxin) has
shown tissue damage in the reticuloendothelial system. The
macrophages in the spleen and liver remove the toxin from
circulation (Vitetta et al, Science, 219, 644-650 (1983)). However,
there is no damage to kidney cells by the immunotoxins used
according to the present invention, showing that the toxin-laden
engineered cells are not toxic. For other engineered red cells the
main target for the contained toxin would be the splenic
macrophages. Since these cells can be replaced by stem cells, any
resulting damage would not be irreversible. Further, experimental
evidence has pointed to the impossibility of exhausting the
reticuloendothelial system because of the replacement of
macrophages from cells in the bone marrow (Van Furth and Cohn, J.
Exp. Med., 128, 415-424 (1968)).
h. Fusion With Target Cells
Since T4 cells, required for proper immune system functioning,
possess the CD4 antigen they will selectively bind to the infected
cells and ultimately be lysed. Lysis can be measured in vitro by
incorporation, into cultured cells, of radioactive moieties, e.g.,
Cr-51. Release of the isotope into the medium above the cells
(monitored using a well-type counter) is a measure of lysis. Such
lysis correlates with the formation of syncytia (as generated by
the fusion of CD4-containing cells with HIV-infected cells). Such
fusion was monitored by the procedure of Example 5, infra, with
results shown in the corresponding Table 1, given hereinbelow.
Erythrocytes into which has been inserted CD4 antigen can be
examined by freeze-etching electron microscopy as well as by thin
section electron microscopy. Briefly, erythrocytes with CD4-antigen
are incubated with mouse monoclonal anti-CD4. After washing, these
cells were incubated with 10 nm gold beads which are coated with
goat anti-mouse IgG. In freeze-fracture replicas, the 10 nm beads
were observed around the circumference of the cross-fractured
erythrocytes. The number of observed beads per cell is dependent on
the chance nature of the fracture and is not necessarily a measure
of the actual number of beads bound to that cell. In the best
cases, the beads appear regularly in a row along one side of a
cross-fractured erythrocyte at intervals of 40 to 50 nm. Due to the
"onion ring" effect produced by etching, it is not possible to
determine exactly the distance of the beads from the erythrocyte
membrane. In thin sections of the same sample, the electron dense
10 nm gold beads are also observed at the membrane surface of the
erythrocyte. Freeze-etching images of CD4-bearing erythrocytes
following incubation with HIV-infected H9 cells showed 100 nm
"irregularities" or extrusions that have altered the distribution
of membrane proteins on the erythrocyte membrane face. This
suggests a fusion of the virus with the erythrocyte surface
(presumably to the CD4 antigen).
Liposomes prepared with CD4 in the bilayers (as per Example 1,
infra) can be examined by freeze-etching electron microscopy and
are found to be both uni-and multilamellar with diameters in the
range of 300 to 500 nm. After incubation of liposomes for 8 hours
with HIV-infected H9 cells, virus particles are found to be
attached to liposomes into which has been inserted the CD4 protein
but not those without this protein. This also shows that the
protein, when inserted into the membrane by the present invention,
is oriented properly. Virus particles can be identified both by
size (about 100 nm) and the presence of membrane proteins.
Liposomes were identified by size and encapsulated dextran.
To demonstrate fusion of CD4-liposomes with infected cells and
delivery of the contents of the liposome to the cell interior, the
following procedure can be employed. CD4-liposomes, encapsulating
ferritin (as per the general procedure of Example 1, infra), are
incubated with HIV-infected H9 cells, or with normal H9 cells, for
8 hours. The cells are then washed and fixed. Liposomes without
CD4, but encapsulating ferritin, are used in like fashion.
Thin-section electron microscopy showed that the
liposome-encapsulated ferritin had been transferred to lipid
droplets in the infected cells. Liposomes were observed inside
large cytoplasmic vacuoles inside the infected cells. Instances of
liposome fusion with the plasma membranes of the cells were also
found. These results were not found where the liposomes did not
possess the CD4 antigen.
CD4-bearing liposomes formed with phosphatidyl ethanolamine
lisamine rhodamine (rhodamine being a fluorescent dye and here
attached to the lipid) in the membrane and encapsulating
F1-dextran, are incubated with HIV-infected H9 cells for 8 hours,
then washed and fixed. A similar procedure is followed using the
same liposomes, but without the CD4 antigen being present in their
bilayers.
Freeze-etching images showed virus particles attached to the
liposomes bearing the CD4 antigen but not to those without it. In
addition, liposomes with the CD4 antigen were shown in the process
of fusing with the HIV-infected H9 cells (i.e., they exhibited
apparent membrane continuities). Liposomes without CD4 antigen
appeared to be resting on the cell membranes but showed no evidence
of actual fusion.
Fluorescence analysis of these cells showed significant rhodamine
fluorescence in experiments where the liposomes had the CD4 antigen
in their bilayers, as well as the lipid-rhodamine conjugate. This
fluorescence was diffuse, as well as punctuate, but in all cases
was excluded from the nucleus of the cell. Diffuse fluorescence
indicates that the liposome has fused with the plasma membrane of
the cell. Punctuate fluorescence can be due to liposomes collecting
on the cell surface but not fusing, although perhaps being in the
early stages of binding and fusing. It can also be due to the
sequestering of liposomal components in digestive compartments of
the cell, as well as to the results of endocytosis of the
liposomes. However, exclusion of the dye from the nucleus indicates
that the cell is still viable.
Where the same experiment employed uninfected healthy H9 cells,
only occasional fluorescence was detected from liposomes adhering
at the cell surface. No significant fluorescence was detected when
liposomes not bearing CD4 antigen were used, regardless of whether
the H9 cells were infected or not.
Because any cells possessing the CD4 antigen will also bind to
infected-cells possessing the viral glycoprotein (gp120), the use
of toxin-laden cells or liposomes (having the CD4 antigen
incorporated into their membranes) permits fusion of these cells or
liposomes with the HIV-infected cells, with subsequent destruction
of the latter before they can bind to, and fuse with, healthy T4
cells. This serves to protect the patient's immune system from
further destruction by the HIV-infected cells, and hopefully at a
time before it has undergone irreparable damage.
Non-limiting examples of toxins that can be advantageously used in
the present invention include ricin (a protein of MW 25,000 whose
toxic A chain is all that is required), abrin (a toxalbumin
obtained from the seeds of certain plants), gelonin (a protein of
MW 23,000 and having the advantage of being non-immunogenic) and
diphtheria toxin. Most of these are commercially available. Gelonin
is prepared by the method of Pihl et. al., J. Biol. Chem., 255,
6947-6953 (1980). The examples of this invention are set out below
are given in terms of using ricin and/or gelonin as the toxin but
any suitable cytotoxic agent can be substituted for these without
any major modification of the basic procedure.
Clinical Use
The modified cells making up this invention can be used to treat
AIDS by selectively eliminating HIV-infected lymphocytes from the
peripheral circulation. The procedure involves inserting the
receptor for the AIDS virus (the CD4 antigen) into autologous red
blood cell membranes. Infected cells express the gp120 fusogenic
protein on their exterior surface and thus bind CD4-containing
cells. In AIDS patients, lymphocytes (normally bearing the CD4
antigen) bind to HIV-infected cells (bearing the gp120 protein) and
this results in spread of the infection until all cells are
infected or dead, thus bringing about general failure of the immune
system. Of course, an HIV-infected lymphocyte will bind to, and
fuse with, any CD4 bearing cell and not just T-helper lymphocytes.
As has been shown, this includes CD4-bearing red cells. Where the
CD4-erythrocyte contains within it a cytotoxic agent, the fusion
results in the death of both cells. In addition, since the virus
itself binds to the CD4 protein, any free virus in the bloodstream
would bind to CD4-bearing erythrocytes and be sequestered within
them. Since erythrocytes lack genetic apparatus, the virus cannot
multiply in them. Thus, there is a two fold effect: sequestering of
the small amount of free virus in the blood stream and, more
importantly, fusion with, and destruction of, gp120-bearing
infected-lymphocytes before they can bind to healthy T-cells and
spread the virus.
In addition to the injection intravenously of engineered red blood
cells, the toxin can be selectively introduced into other tissues
by use of liposomes. For example, infected cells present in lymph
nodes can be selectively attacked by interstitial injection (e.g.,
between the fingers) of liposomes (bearing the CD4 antigen in their
bilayers and containing gelonin, ricin or some other toxin
encapsulated within the liposome) into the lymph nodes. An
advantage of such method of treatment is that the liposomes
generated by the methods here presented are of a size approaching
that of small cells and thus would not be degraded by endocytosis
following injection.
In addition, the CD4-bearing cells and liposomes making up this
invention can be employed as part of an in vitro assay fro
detecting the presence of gp120-bearing (i.e., infected)
lymphocytes (employing the procedure of Example 5 below). Such a
procedure involves the production of cells or liposomes according
to this invention. The cells or liposomes making up the diagnostic
reagent have CD4 antigen in their membranes, contain a cytotoxic
agent in their cytoplasm, and are labeled with a radioactive
substance, advantageously chromium-51. Because HIV-infected cells
bear the gp120 virus protein in their membranes, such cells would
fuse with the CD4-bearing, toxin containing, radiolabeled cells
with subsequent release of the radiolabel into the surrounding
medium.
To use this invention as a diagnostic reagent, blood is withdrawn
from a patient, optionally one suspected of having AIDS, the white
cells (especially the lymphocytes) are collected by standard
procedures (for example, those already described in this
application) and a small aliquot is mixed in vitro with an aliquot
of the cells or liposomes of this invention, as described in
Example 5, below, wherein liposomes are used for demonstrative
purposes. After incubation at or around 37.degree. C. for a
sufficient period of time for cell fusion to occur, optimally up to
24 hours, a smaple of the cell medium is collected and the
radioactivity measured. The measurement is duplicated using white
cells from the blood of a normal person as a control. Detection of
an elevated level of radioactivity (following calculation via a
corrective formula) in the medium surrounding the cells taken from
the patient relative to the control cells indicates fusion and,
thereby, the presence in the patient's blood of cells containing
the gp120 protein. The latter is interpreted to mean that said
cells are infected with the AIDS virus and that the patient
therefore has AIDS. Since antigens other than CD4 can be introduced
into the membranes of the cells or liposomes of this invention,
other viral diseases could be diagnosed using this procedure.
Heretofore, diagnostic procedures for AIDS have commonly relied on
detection of the presence of anti-HIV antibodies in the blood of
patients suspected of having AIDS. However, such a finding does not
necessarily indicate the occurrence of the disease but merely that
the patient has been exposed to the virus or some of its antigens.
The diagnostic procedure disclosed herein has the advantage of
indicating the presence of infected cells in which the virus is
replicating, i.e., an active infection. Applicants intend to rely
on all equivalents thereof.
The cells and liposomes making up the present invention can be
further utilized as a means of delivering a therapeutic amount of
anti-viral drugs directly to cells such as macrophages and Kuppfer
cells. The latter types of cells (part of the reticuloendothelial
system) do not possess receptors for HIV. However, HIV-infected
cells express foreign antigen on their surfaces and are eventually
taken up and phagocytozed by macrophages and Kuppfer cells. It is
believed that this route is used by the virus to infect cells of
the reticuloendothelial system despite these cells'of CD4 antigen
on their surfaces. To protect these cells from harboring the virus
and allowing its replication, the cells and liposomes of the
present invention can be used to deliver a therapeutic amount of an
anti-AIDS drug directly to macrophages, Kuppfer cells and other
cells of the reticuloendothelial system. This has the effect of
protecting these phagocytic cells from becoming infected with the
virus following their ingestion of infected cells and other virus
contaminated debris.
By way of example, such anti-AIDS chemotherapeutic agents as AZT
(azido-3'-deoxythymidine), ribavarin and DDC are readily
encapsulated within the cells and liposomes of the present
invention. This is most easily accomplished by using the
encapsulation procedure described in U.S. Pat. No. 4,652,449,
issued Mar. 24, 1987, the entire disclosure of which is
specifically incorporated herein. Either before or after the
encapsulation of the therapeutic agent, the procedures of the
present invention are used to insert the appropriate antigenic
protein (CD4 where AIDS is the disease to be treated) into the
membrane of the erythrocytes and liposomes. The result is an
erythrocyte or liposome containing therein a therapeutic quantity
of an active anti-AIDS drug and which has incorporated into its
membrane the CD4 antigen to serve in directing it to, and inducing
fusion with a cell infected with the AIDS virus. Following fusion,
this fused cellular complex is recognized as foreign (because of
the virus-coded proteins in the membrane of the infected cell or
cells) and phagocytozed by macrophages and other
reticuloendothelial cells. As a result, the anti-AIDS drug is
introduced directly into the phagocytic cells and prevents viral
replication in those cells, thus closing off another avenue by
which the virus can replicate itself.
The advantages of such a procedure are that the drug is not free in
the blood stream and thus is not available to cause any undesirable
and deleterious side effects. In addition, because the antigen in
the membrane of the drug carrying cell or liposomes is specific for
cells carrying the gp120 protein in their membranes (i.e.,
HIV-infected cells), healthy cells will not be exposed to the drug
and thus any inherent toxicity of the drug will be greatly reduced.
The net result is that the overall dosage of the drug can be
reduced, thus reducing overall cost, while the effective
therapeutic plasma concentration of the drug will also be increased
(because it is not needlessly spread throughout the body). A
further advantage is that, because the drug is effectively
sequestered within the antigenically modified erythrocytes and
liposomes, it is unable to spread throughout the tissue fluids of
the body (where it is less likely that there will be any
virus-infected cells).
The invention is now described with reference to the following
non-limiting examples.
EXAMPLES
Example 1
Insertion of the human leukocyte antigen CD4 into the Erythrocyte
Plasma Membrane
a. A typical preparation of liposomes was performed as follows: the
lipids to be used (which were stored prior to use at -30.degree. C.
in 2:1 (v/v) chloroform/methanol) were mixed in different
proportions. The most common procedure involved mixing phosphatidyl
ethanolamine (purchased from Sigma Chemical Co., St. Louis Mo., and
purified according to Singleton et al., J. Am. Oil Chem. Soc., Vol.
42, 53-61 (1965)), phosphatidyl choline (Sigma Chemical Co.),
phosphatidyl serine (Sigma) and cholesterol (Sigma) in the molar
ratio of 1:2:1:1.5, respectively, and typically a total weight of 5
mg was used. This mixture (final concentration 10 to 30 mM) was
dried to a thin film under a stream of nitrogen, then in vauco for
1 hour (to remove residual traces of organic solvent). The
liposomes were prepared by reverse phase evaporation (see Szoka,
Proc Nat Acad Sci U.S.A., Vol 75, 4194-4198 (1978)). Briefly, the
lipid material was dissolved in 4.5 ml of freshly distilled ether
and sonicated 15 seconds in a bath type sonicator with 1.5 ml of
phosphate buffered saline (PBS). Solubilization was aided by the
addition of n-octyl-D-glucopyranoside (OG) so that the
detergent/lipid molar ratio was 8:1. The ether was removed under
reduced pressure in a rotary evaporator and the liposome suspension
was diluted to 4.5 ml with PBS or else in borate buffer, pH 7.2.
Alternatively, the liposomes can be generated by the procedure
described by Philippot et al., Biochim. Biophys. Acta, 734, 137-143
(1983), which employs adsorption on hydrophobic beads.
b. For reconstitution, a suspension of liposomes (containing 5 mg
of lipid in 1 ml of borate buffer, pH 7.2) was mixed with a
solution containing the proteins for incorporation. The puriffied
CD4 (at a concentration of from 4 to 8 mg protein/ml in PBS
containing 12 OG) was added to the lipid-detergent mixture. The
ratio of lipid to protein, by weight, was maintained between 5 and
10. Dimethylsuberimidate (purchased from Sigma) was added slowly at
a temperature of 10.degree. C. until a final suberimidate
concentration of 1.5 mg/ml was attained. The mixture was then
incubated for 30 minutes at a temperature of 10.degree. C. and then
dialysed against borate buffer for 2 hours at a temperature of
4.degree. C. The resulting mixture was then chromatographed on a
Sepharose 4B column in order to separate liposomes from unreacted
proteins.
This procedure yields about 5,000 to 20,000 molecules of CD4
antigen per liposome. The liposomes were characterized using flow
cytometry and freeze fracture. Their internal volume was found to
be between 12 and 16 liters per mole of lipid and their average
external diameter was found to be about 450 nm.
In addition to reconstituting with pure CD4, the procedure was also
carried out using a quantity of lysozyme equal to that of CD4 as
part of the protein mixture. This is then incorporated along with
the CD4 and has the advantage of preventing fusion of liposomes
with each other during the later fusion steps that produce modified
erythrocytes.
The enzymatic activity of the lysozyme (if lysozyme is used) can be
assayed by the Micrococcus leisodeikticus assay (Arvinte et al,
Proc. Natl. Acad. Sci. USA, 83, 962-966 (1986)).
c. A volume of fresh whole blood was diluted with the same volume
of phosphate buffered saline (PBS, 5 mM phosphate, 145 mM NaCl, pH
7.4, and separated from plasma by centrifugation (640 g for 30
minutes at 4.degree. C.). Both the supernatant and the buffy coat
of white cells were discarded. Polymorphonuclear leukocytes were
removed by absorbent cotton filtration. The erythrocytes are then
resuspended in PBS, pH 7.4, followed by 3 more washes (each with
centrifugation at 2000 rpm for 30 minutes at 4.degree. C.).
d. The hematocrit was adjusted to 70% and the erythrocytes (about
5-10 million) were then incubated with an equal volume of liposome
suspension (with or without lysozyme and using sufficient liposomes
to give a ratio of 1 to 10 liposomes per red cell) and 1.4 m) of
sodium acetate buffer (0.02M sodium acetate/0.145M NaCl) at final
pH 5.5 for 30 minutes at a temperature of 37.degree. C. The
erythrocytes were then collected by centrifugation at 1400 g for 20
min at 20.degree. C. Immunofluorescence assay with
fluorescent-labeled anti-CD4 antibodies was used to quantitatively
measure the presence of CD4 antigen in the plasma membranes of a
small sample of the erythrocte/liposome hybrids.
Alternatively, CD4 can be inserted without the use of liposomes.
Here, 1.5 mL of sodium acetate buffer (0.02N, pH 4.7, 0.145M NaCl),
60 microliters of a solution containing 0.1 to 0.4 mg of CD4 (in 1%
octylglucoside) and 30 microliters of red blood cell suspension
(produced by combining 50 microliters RBC pellet with 1 mL PBS, pH
7.4) are mixed in an Eppendorf centrifuge tube (15 mL size was
convenient) and incubated at 37.degree. C. for 60 to 90 seconds.
The above 3 solutions (buffer, protein and RBC) should be warmed to
37.degree. C. prior to mixing. After incubation, 10 mL of PBS, pH
7.4, was added (to halt exposure of cells to low pH) and the cells
were then concentrated by centrifugation of the reaction mixture at
3000 rpm for 4 min in the fixed angle rotor of an Eppendorf
centrifuge. The supernatant was removed and the cells then
resuspended in PBS, pH 7.4. Three additional washes were performed
in PBS, under the same conditions.
Example 2
Measurement of Incorporated CD4 Antigen
The presence of antigenic activity of the incorporated CD4 antigen
was measured using fluoresccin-isothiocyanate labeled anti-CD4
antibodies by the procedure of fluorescence-activated cell sorting
(using an Epics V cell sorter from Coulter). For this
determination, 2 samples were used:
Sample A: Red blood cells into which CD4 molecules had been
incorporated.
Sample B: Red blood cells containing no CD4 protein.
In the following description, FITC refers to fluorescein
isothiocyanate, a fluorescent label for protein chains.
The procedure used was as follows:
Both samples, A and B, were washed with PBS, pH 7.4, and
concentrated by centrifugation (at 3000 rpm for 4 minutes in an
Eppendorf centrifuge). Over the cell pellets, each about 10
microliters, there was layered, in each experiment, a solution of
one of the following monoclonal antibodies:
1. 10 microliters of "Anti-T4-FITC" (Pel-Freez monoclonal antibody,
M 102-10-OAX, Brown Deer, Wis. 53223).
2. 10 microliters "Leu-3a-Pe" (Anti-human Leu-3a Phycoerythrin
conjugate from Becton Dickinson, Mountain View, Calif. 94039).
3. 10 microliters ("OKT-4A-FITC" (Ortho-mune OKT-4a Murine
monoclonal antibody-FITC conjugate, Anti-human inducer/helper T
Cell, from Ortho Diagnostic Systems, Inc., Raritan, N.J.
08869).
The suspension was agitated to mix the cell mass with the antibody
solutions. The cells were allowed to react with the fluorescence
labeled anti-CD4 antibodies for 15 minutes at 22.degree. C.
After incubation, 1 ml of PBS, pH 7.4, was added to the 2 samples
and the cells suspensions again concentrated by centrifugation (as
above). The supernatants (A and B) contained the antibodies that
did not bind to the cells and these were removed. The process of
washing with PBS and concentrating by centrifugation was repeated
twice with removal of each of the supernatants.
The cells were suspended in PBS and examined. Only the cells from
sample A were fluorescent by microscopy, spectrophotometry and FACS
assay, so that only they could be considered to have incorporated
the CD4 antigen into their membranes.
In addition, the amount of fluoresent material (FITC) in the pooled
supernatants was measured by fluorescence spectroscopy using an
excitation wavelength of 470 nm and an emission wavelength
detection at between 480 and 600 nm.
Protein fluorescence measurements utilized an excitation wavelength
of 280 nm with the same emission range as above.
The difference in the fluoresent intensities between the pooled
supernatants, A and B, was directly proportional to the amount of
fluorescent label bound to the erythrocytes. Therefore, knowing the
initial antibody concentration and the number of red blood cells,
it was simple to calculate the mean value for the number of CD4
molecules per cell.
In the above procedure, each sample, A and B, contained about 14
million cells and the total amount of fluorescent monoclonal
antibodies was about 0.0021 mg. The average molecular weight of CD4
antigen is about 58,000 daltons so that each incubation mixture
contained roughly 7 trillion antibody molecules. Using these values
where
and
and from the protein fluorescence spectra (excitation=280 nm) the
value calculated was R=1.1. The corresponding value for FITC
fluorescence was R=1.33.
Assuming that the CD4 molecules in the cell membranes were
saturated by the antibodies, the number of bound antibodies equals
the number of incorporated CD4 molecules (N in the above formula).
Using R=1.1, the value for N was 37,000. Using R=1.33, the value
was N=59,300. The value used depends on whether you measure protein
fluorescence or protein-bound FITC fluorescence and the result is
therefore a means of these values. Therefore, our calculations
showed between 37,000 and 60,000 CD4 molecules per cell.
Example 3
Encapsulation of Ricin Toxin in Erythrocytes
A. Erythrocytes prepared in accordance with the procedure given in
Example 1 were washed several times with chilled 0.15M NaCl and
centrifuged to give a pellet. The cells were then resuspended in
PBS at pH 7.4.
b. The suspension of erythrocytes was then washed with a solution
containing up to 0.1 mM of pure ricin toxin (purified A chain from
Sigma Chemical Co.) in PBS, pH 7.4. The erythrocytes were
centrifuged at 1000 g for 10 minutes, the supernatant decanted and
the final hematocrit adjusted to 70% with saline.
c. The erythrocyte suspension was cooled to 4.degree. C. and
allowed to flow continuously into the blood compartment of a
conventional hemodialyzer having a dialysis surface of 0.41 square
meters and a membrane thickness of 13.4 microns. Constant
erythrocyte flow rate of 20 to 60 ml/minute was maintained with a
peristaltic pump. The hemodialyzer was fed at a constant flow rate
of 500 ml/minute with a low ionic strength buffer (0.01M sodium
phosphate, 0.01M sodium bicarbonate, 0.002M glucose) at pH 7.4 and
temperature maintained at 4.degree. C. During this dialysis step
the erythrocytes were lysec and collected at 37.degree. C. before
being resealed through the addition of a tenth volume of a
hypertonic solution containing (per liter) 1M chloride salt with a
K to Na ratio of 8.3 (in order to maintain a high ATP content in
the resealed cells).
d. The cell suspension was then collected and maintained at
37.degree. C. for 30 minutes to permit resealing of the cells. The
erythrocytes are then washed twice with a 0.15M NaCl solution
containing (per liter) 1 mM calcium chloride, 1 mM magnesium
chloride, and 2 mM glucose. The erythrocytes are then suspended the
native autologous plasma before infusion at a chosen
hematocrit.
Example 4
Alternative Procedure for Encapsulation of Ricin or Gelonin Toxins
into Erythrocytes
a. Beginning with the same lipid mixture of example 1 and taking it
through the evaporation of residual organic solvents, the toxic
material (e.g., ricin, gelonin, etc) to be encapsulated was
introduced in HEPES buffer (10 mM HEPES (pH7.4)/1 mM EGTA/150 mM
NaCl) as per the procedure described by Philippot et al. Sufficient
toxin was used to give a final value of about 0.01 to 0.02
micrograms per billion liposomes. The two phase system was vortexed
briefly and the lipids were hydrated 30 minutes at a temperature
above the highest transition temperature of the components in the
mixture. Small amounts of detergent (Triton X-100) were added and
the volume of the samples was adjusted to 0.625 ml with HEPES
buffer. After vigorous shaking the detergent was removed.
b. Three different techniques were used to remove detergent:
i. The sample was placed in a dialysis bag 1 cm wide and dialyzed
against 1 liter of 0.01M Tris-MCl (pH 7.4)/1 mM EDTA/0.15M NaCl
with 4 changes of the medium.
ii. The volume of dialysis medium was reduced to 100 ml and
Bio-Beads (type SM-2 from Bio-Rad, Richmond, Calif.) were added
outside the bag in the buffered medium. The medium was not
changed.
iii. In some experiments the Bio-Beads were added directly to the
liposome preparation in a test tube, and placed on a rotary mixer
running at 10 RPM for at least 3 hours.
c. When necessary, the liposome suspension was passed through a
Sepharose 4B column to remove the non-encapsulated material. The
ricin containing liposomes are then used for the insertion of
lysozyme and CD4 as in Example 1.
d. The erythrocytes are then incubated with the ricin- (or
gelonin-) containing liposimes as described in Example 1. The
fusion efficiency was monitored by fluoescence microscopy and by
FACS analysis. For larger scale preparations the procedure can be
carried out using ricin without the fluorescein label since the
latter was needed only for monitoring purposes.
Example 5
In Vitro Interaction of HIV-Invected Cells with CD4-Liposomes
Containing Gelonin
a. A T-cell population, H9, was cloned from the HT cell line and
some of the cells were persistently infected with HIV isolate. The
cells are then cultured in RPMI 1640 medium (containing 10%
decomplemented fetal calf serum, 0.2 mM glutamine) as described in
Yoffe et al., Proc. Nat. Acad Sci, U.S.A., 84, 1429-1433 (1987).
H9/HIV cells are morphologically indistinguishable from uninfected
cells when examined by light microscopy. Thus, virus production by
the H9/HIV cells was monitored by electron microscopy, as well as a
standard reverse transcriptase assay.
b. Cells containing the CD4 antigen were obtained from blood
donors, some of whom were negative for anti-HIV antibody and some
of whom were positive (as described in Yoffe et al., supra).
c. The cells were labeled using 0.5 mCi of (Cr-51)-sodium chromate
(from New England Nuclear, Boston, Mass.) for 90 minutes at
37.degree. C. and then washed three times with phosphate-buffered
saline. The labeled cells were then plated into culture dishes at a
concentration of 10,000 cells per well.
d. CD4-liposomes, CD4-liposomes plus free gelonin, and
CD4-liposomes encapsulating gelonin were each added to separate
wells containing T-cells at a ratio of 5 liposomes per T-cell. Both
non-infected H9 cells and infected HIV/H9 cells are then incubated
separately with each of the liposome preparations for 18 hours at
37.degree. C.
e. After incubation, a 100 microliter aliquot of the supernatant
fluid from the cultures was collected and the radioactivity was
measured by liquid acintillation. For each run the experiment was
done in triplicate (i.e., 3 cultures are used for each run). The
results are interpreted in terms of the percent specific
chromium-51 release (SP REL) which indicates the extent of cell
fusion and is defined by the formula: ##EQU1## where
ER=experimental release
MR=a maximal release
SR=spontaneous release
The spontaneous release (SR) was measured by harvesting a 0.1 ml
aliquot from the supernatant fluid in wells containing only labeled
cells (i.e., H9 or HIV/H9).
Maximal release was measured by removing 0.1 ml of supernatant
fluid from labeled cells lysed with 0.1 ml of 1% Triton X-100.
Radioactivity was measured using a well counter.
f. Following the 18 hour incubation both the H9 cells and the
HIV/H9 cells were harvested, washed and stained by conventional
procedures with trypan blue to determine the extent of
survival.
Significant amounts of Cr-51 release were observed only in
experiments where HIV-infected H9 cells were exposed to liposomes
containing both CD4 antigen and gelonin and not where only
CD4-liposomes were used or where the gelonin was free in the medium
and not encapsulated within the CD4-liposome. Results for this
experiment are shown in Table 1 hereinbelow.
TABLE 1 ______________________________________ Cr-51 release
following fusion of engineered liposomes with infected and normal
H9 cells WELL # Cells Liposomes Prep. Spec. Rel.
______________________________________ 1 H9 -- 2800 cpm 2 H9
CD4-liposomes 2800 cpm 3 H9 CD4-liposomes + 2800 cpm free gelonin 4
H9 CD4-liposomes 2800 cpm (containing gelonin) 5 HIV/H9 -- 2800 cpm
6 HIV/H9 CD4-liposomes 2800 cpm 7 HIV/H9 CD4-liposomes + 2800 cpm
free gelonin 8 HIV/H9 CD4-liposomes 5700 cpm (containing gelonin)
______________________________________ # Specific release given as
cpm per 10,000 cells following an 18 hour incubation at 37.degree.
C. as per Example 5. *# free gelonin concentration was 0.02 mg per
10.sup.11 liposomes.
Example 6
Clinical Treatment of Anti-HIV Positive Patients With Engineered
Erythrocytes
A human patient who has tested positive for anti-HIV antibodies is
treated with engineered red cells containing ricin, abrin, gelonin
or diphtheria toxin as follows. The patient is injected
intravenously (e.g., in the arm) with up to 20 ml of a saline
suspension of packed engineered red cells (wherein virtually all of
the cells contain the CD4 antigen and cytotoxin). The patient's
condition is monitored by measuring the level of anti-HIV
antibodies in the circulation as well as the general progress of
his condition. The initial injection can be followed up by
additional injections as the physician deems warranted.
Example 7
Clinical Treatment of Patients With ARC Using Liposomes and
Engineered Erythrocytes
Human patients believed to have ARC (AIDS Related Complex) are
treated for the condition in much the same way as in Example 6.
However, here the patients are injected with a combination of
engineered red cells and liposomes (both containing CD4 in their
membranes and cytotoxin. In this disease condition the main target
is the lymph nodes so that advantageously the patient is injected
interstitially (e.g., between the fingers) with a mixture of about
100 billion liposomes and an optimally active amount of modified
erythrocytes. The patient's condition is monitored as in Example 6
and additional treatments given as needed.
Example 8
Clinical Treatment of Anti-HIV Positive Patients With Engineered
Erythrocytes Containing AZT
A human patient who has tested positive for anti-HIV antibodies or
by the diagnostic procedure disclosed in this application is
treated with the antigenically modified red cells or liposomes of
the present invention containing azido-3'-deoxythymidine (AZT) as
follows. The patient is injected intravenously (e.g., in the arm)
with up to 20 ml of a saline suspension of packed red cells or
liposomes (wherein virtually all of the cells or liposomes contain
the CD4 antigen and a cytoplasmically sequestered therapeutic
amount of AZT). Such treatment is adjusted by the clinician so that
the total dosage of the drug is kept within safe limits, optimally
between 100 to 300 mg per 4 to 6 hour period. The patient's
condition is then monitored by measuring the presence of anti-HIV
antibodies in the circulation (or via the diagnostic procedure set
forth in the application) as well as the general progress of his
condition. The initial injection can then be followed up by
additional injections every 4 to 6 hours as the attending physician
deems warranted.
It is understood that the specification and claims are illustrative
but not limitative of the present invention and that other
embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *